NET and SERT[1]

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method was developed for the simultaneous determination of six synthetic adulterants, namely fenfluramine, phenolphthalein, N-di-desmethyl sibutramine, N-mono-desmethyl sibutramine, sibutramine, and orlistat. The method was applied to the analysis of herbal weight-reducing dietary supplements. Chromatographic separation of the analytes on a C(8) reversed-phase column was achieved using a gradient elution of solvent A: acetonitrile and solvent B: aqueous 20 mM ammonium formate solution. Sildenafil was utilized as an internal standard for quantification. The MS detector was operated in positive electrospray ionization mode. Selected-ion monitoring (SIM) was carried out for m/z 232, 319, 252, 266, 280, 496, and 475 for fenfluramine, phenolphthalein, N-di-desmethyl sibutramine, N-mono-desmethyl sibutramine, sibutramine, orlistat, and sildenafil, respectively. The method was validated for accuracy, precision, linearity, and selectivity. The limits of detection for the six synthetic adulterants ranged from 0.0018 to 0.73 microg g(-1). The proposed method was used for a small survey of 22 dietary supplements of which eleven samples were adulterated with phenolphthalein, N-mono-desmethyl sibutramine, and sibutramine at levels from 0.212 to 96.2 mg g(-1)[3].

Sibutramine is a centrally acting monoamine reuptake inhibitor prescribed as an appetite suppressant in the management of obesity. Its effects are mostly attributable to serotonin and norepinephrine transporter (SERT and NET, respectively) inhibition by its potent metabolites mono-desmethylsibutramine (M1) and di-desmethylsibutramine (M2). However, there is a paucity of in vivo data in humans about mechanisms underlying both clinical efficacy and the dose-independent non-response observed in a minority of patients. Twelve healthy male patients (mean age 41 years) completed a double-blind, placebo-controlled, within-subject crossover investigation of brain SERT occupancy by sibutramine 15 mg daily at steady state. Correlations were measured between occupancy and (i) plasma concentrations of sibutramine, M1 and M2; (ii) appetite suppression. (11)C-DASB PET scans were performed on the HRRT camera. Binding potentials (BP(ND)) were calculated by the Logan reference tissue (cerebellum) method. SERT occupancy was modest (mean 30+/-10%), was similar across brain regions, but varied widely across subjects (15-46%). Occupancy was correlated positively (p=0.09) with M2 concentration, but not with sibutramine or M1. No significant appetite suppression was seen at <25% occupancy and greatest suppression was associated with highest occupancy (25-46%). However, several subjects with occupancy (36-39%) in the higher range had no appetite suppression. SERT occupancy by clinical doses of sibutramine is of modest magnitude and may be mediated predominantly by M2 in humans. 5-HT reuptake inhibition may be necessary but is not sufficient for sibutramine's efficacy in humans, supporting preclinical data suggesting that the hypophagic effect requires the co-inhibition of both SERT and NET[1].

Metabolism / Metabolites
Desmethylsibutramine is a known human metabolite of (S)-sibutramine.

[1]. Brain serotonin transporter occupancy by oral sibutramine dosed to steady state: a PET study using (11)C-DASB in healthy humans. Neuropsychopharmacology. 2010 Feb;35(3):741-51.

[2]. The contribution of metabolites to the rapid and potent down-regulation of rat cortical beta-adrenoceptors by the putative antidepressant sibutramine hydrochloride. Neuropharmacology. 1989 Feb;28(2):129-34.

[3]. Analysis of six synthetic adulterants in herbal weight-reducing dietary supplements by LC electrospray ionization-MS. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2008 Jul;25(7):822-30.

[4]. Quantification of sibutramine and its two metabolites in human plasma by LC-ESI-MS/MS and its application in a bioequivalence study. J Pharm Anal. 2012 Aug;2(4):249-257.

Sibutramine hydrochloride is a monoamine reuptake inhibitor, and its pharmacological characterization in rodents suggests antidepressant activity. The tertiary amine metabolites of sibutramine hydrochloride—the secondary amine (BTS 54 354) and primary amine (BTS 54 505)—have similar in vivo pharmacological activities to the parent compound. Therefore, each compound showed potent activity in an acute behavioral model predicting antidepressant effects and comparable ability to inhibit monoamine uptake in vivo. Furthermore, BTS 54 354 and BTS 54 505, like sibutramine hydrochloride, rapidly and effectively downregulated β-adrenergic receptors in the rat cortex. However, in vitro, the secondary and primary amines exhibit significantly higher activity as inhibitors of norepinephrine, dopamine, and serotonin uptake than sibutramine hydrochloride. The potent inhibition of norepinephrine reuptake by secondary and primary amine metabolites may be one of the reasons why the potential antidepressant sibutramine hydrochloride induces rapid and potent downregulation of β-adrenergic receptors in rats. [2] Obesity can be considered an epidemic chronic disease, and its incidence has increased exponentially in recent years. Anti-obesity drugs such as sibutramine have been helpful to some extent. However, quantitative analysis of such drugs in biological samples is often quite difficult. This report develops and validates a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of sibutramine (SB) and its two metabolites N-desmethylsibutramine (DSB) and N-didesmethylsibutramine (DDSB) in human plasma. Chromatographic separation of SB, DSB, and DDSB was performed using a Zorbax SB-C18 (4.6 mm × 75 mm, 3.5 μm, 80 Å) analytical column and a 5 mM ammonium formate:acetonitrile (10:90, v/v) mobile phase. Multiple reaction monitoring (MRM) in positive ion mode was used to detect SB, DSB, and DDSB at m/z values of 280.3/124.9, 266.3/125.3, and 252.2/124.9, respectively. Analytes and internal standards were extracted from human plasma using liquid-liquid extraction. This method was validated for SB, DSB, and DDSB within a linear concentration range of 10.0–10,000.0 pg/mL, with a correlation coefficient (r) ≥0.9997. The drugs and their two metabolites were stable in plasma samples. The validated method has been successfully applied to bioequivalence and pharmacokinetic studies in fasting human volunteers. [4]

C16H24CLN

265.82

265.16

168835-59-4

Desmethyl Sibutramine hydrochloride;84467-94-7

10199199

White to off-white solid powder

4.786

1

1

5

18

252

0

PLXKZKLXYHLWHR-UHFFFAOYSA-N

InChI=1S/C16H24ClN/c1-12(2)11-15(18-3)16(9-4-10-16)13-5-7-14(17)8-6-13/h5-8,12,15,18H,4,9-11H2,1-3H3

1-[1-(4-chlorophenyl)cyclobutyl]-N,3-dimethylbutan-1-amine

Desmethylsibutramine; Desmethyl Sibutramine; 168835-59-4; N-Desmethylsibutramine; 1-[1-(4-chlorophenyl)cyclobutyl]-N,3-dimethylbutan-1-amine; 889I657R9P; {1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutyl}(methyl)amine; Desmethyl Sibutramine (hydrochloride);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

 (Please use freshly prepared in vivo formulations for optimal results.)